Phylogenetic relationship and characterization of the complete mitochondrial genome sequence of Opsarius caudiocellatus (Cypriniformes: Danionidae: Chedrinae)

Abstract In this paper, we first report the complete mtDNA sequence of Opsarius caudiocellatus with the main aim of providing a basis for further researches of this species. Assembly circular mitogenome was 16,534 bp long encoded 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and one control region. The gene nucleotide composition was estimated to be 28.1% A, 25.2% T, 18.6% G, 28.1% C. A maximum-likelihood (ML) phylogenetic tree was reconstructed using the 13 concatenated mitochondrial protein-coding genes of O. caudiocellatus and other 19 species of the subfamily Chedrinae. Result of the phylogenetic analysis revealed that O. caudiocellatus was well grouped with Barilius barila. This study could enrich genetic resources and be helpful to studies on evolution and conservation genetics for O. caudiocellatus.


Introduction
Opsarius caudiocellatus (Chu 1984; Cypriniformes: Danionidae: Chedrinae) is an endemic small freshwater cyprinid fish ($111 mm), which inhabits medium to fast flowing rivers or mountain streams with rich dissolved oxygen, medium to low slope, the substrate of gravel, cobbles and large pebbles. It is mainly distributed in the Nujiang River and Lancang River in China, and it is also distributed in India, Thailand, Vietnam, and Laos. In the past, Barilius caudiocellatus was treated as the synonym name of O. caudiocellatus. The main morphological features of O. caudiocellatus are similar to those of Barilius barila such as dorsal fin iii, 7-8; anal fin ii, 9-11; pectoral fin i, 11-12; ventral fin i, 7, anal fin origin opposite the 4th-7th branching dorsal fin. O. caudiocellatus can be distinguished from Barilius barila as follows: black spots at the base of the caudal fin for O. caudiocellatus not for B. barila, and the black area is positioned in the middle of the dorsal fin for O. caudiocellatus, while on both sides of B. barila ( Figure 1) (Chu 1984). The complete mitochondrial genome is regularly used in detailed distribution surveys which are urgently required for this species, and thus could more precisely reveal the taxonomic status (Qin et al. 2019

Sample collection and preservation
The specimens were collected by gill nets from Nujiang River in Lushui County, Yunnan Province of China (25 84 0 75.91 00 N, 98 86 0 69.44 00 E) in May 2021, under the permission granted by Jiangsu Agri-animal Husbandry Vocational College (NSF2021ZR14). Research involving laboratory animals followed the ARRIVE guidelines (https://arriveguidelines.org/). The dead fish were selected while the live fish were released into the river. Samples were fixed in 95% ethanol and stored at the Aquatic Science and Technology Institution Herbarium (https://www.jsahvc.edu.cn/, Voucher number ASTIH-21b1108d30, Chen Xiao Jiang, 2007020030@jsahvc.edu.cn). According to the morphological characteristics description of O. caudiocellatus by Chu (1984), with tools such as vernier calipers and an anatomical microscope, we identified the species by morphometry and meristematic counts.

Mitochondrial genome sequencing
In this paper, high-throughput sequencing technology was used. Genomic DNA was extracted from a specimen muscle using the Tguide Cell/tissue genomic DNA Extraction Kit (OSR-M401) (Tiangen, Beijing, China). The main experimental steps are as follows: (1) DNA sample quality control; (2) DNA Library Construction; (3) PCR Amplification; (4) Size Selection; (5) Library Quality Check; (6) Library Pooling and Sequencing, the amplified original library was submitted to Illumina HiSeq 4000 Sequencing platform (Illumina, CA, USA).

Assembly, annotation, and analysis
The quality check process was conducted on FastQC Version 0.11.8 (Andrews 2015) to obtain clean data, and the mitogenome was assembled from the clean data (2.46 GB) by MetaSPAdes 3.13.0 (Nurk et al. 2017) with Barilius malabaricus MN650735.1 as reference (Prabhu et al. 2020). The resulting circular contig consensus sequence was annotated and verified with MITOS WebServer (http://mitos.bioinf.uni-leipzig.de/ index.py) (Bernt et al. 2013). Genome Map was drawn by OGDRAW (https://chlorobox.mpimp-golm.mpg.de/OGDraw. html) (Greiner et al. 2019). MEGA X was used for alignments, Figure 2. Mitochondrial genome map of Opsarius caudiocellatus. The arrows represents direction of transcription, H-strand is located in the outer ring and L-strand is located in the inner ring.
analyses, model calculation, and phylogeny reconstruction (Kumar et al. 2018). Maximum-likelihood (ML) phylogenetic tree was reconstructed using the concatenated mitochondrial protein-coding genes of O. caudiocellatus and 19 published species. Rasbora lateristriata (Kusuma and Kumazawa 2016), and Rasbora steineri (Chang et al. 2013) were used as outgroups to root the tree. The best evolutionary model obtained the lowest Bayesian information standard scores (Nei and Kumar 2000).

Conclusions
The complete mitochondrial genome of O. caudiocellatus was sequenced and annotated via high-throughput sequencing technology. The assembly circular mitogenome was 16,534 bp long. The phylogenetic analysis results supported that O. caudiocellatus was clustered together with B. barila. It is suggested that Barilius barila belongs to Opsarius, while Opsarius canarensis belongs to Barilius. This requires more data for further analysis and confirmation. The fundamental genetic data presented here could be beneficial for further genetic and evolutionary researches on O. caudiocellatus.

Ethical approval
Experiments were performed in accordance with the requirement of the Ethics Committee for Animal Experiments of Jiangsu Agri-animal Husbandry Vocational College. These policies were enacted according to the Chinese Association for the Laboratory Animal Sciences and the Institutional Animal Care and Use Committee (IACUC) protocols.